Tyrosine kinase inhibitors combined with chemotherapy significantly improved outcomes in adult Philadelphia- chromosome-positive (Ph+) B-cell Acute Lymphoblastic Leukemia (B-ALL). However, high relapse rate due development of TKI resistance or chemotherapy-induced adverse effects remain the major therapeutic challenges. Furthermore, all TKIs are not effective against Ph+ leukemia-initiating cells (LICs). The tumor suppressor protein phosphatase 2A (PP2A) is inactive in almost all solid and hematopoietic tumors. PP2A inhibition correlates with poor outcome and disease progression, and largely relies on the aberrant expression of CIP2A, SET and/or SETBP1. SETBP1 was discovered as a SET-interacting protein, and recently described as mutated or overexpressed in several myeloid malignancies where it acts as an independent negative prognostic factor and as an inhibitor of PP2A. Thus it is possible that SETBP1 regulates survival and self- renewal of Ph+ B-ALL LICs through inhibition of PP2A. Published work and preliminary data indicate that: SET- dependent PP2A inhibition increases in Ph+ (CML and B-ALL) progenitors and quiescent TKI-resistant CML LICs, respectively; SET downregulation or pharmacologic (i.e. SET-interacting PP2A-activating drugs; PADs) restoration of PP2A activity strongly impaired malignant but not normal hematopoiesis; SETBP1 is induced in an imatinib (IM)-insensitive manner and essential for PP2A inhibition and clonogenic potential of Ph+ B-ALL cells; and a SETBP1-SET/CIP2A complex may exist in Ph+ cells, suggesting that SETBP1 might serve to recruit SET and CIP2A to suppress PP2A activity. Based on these considerations and on the fact that SETBP1 stabilizes SET and augments PP2A inhibition, and ectopic SETBP1 expression confers self-renewal to mouse myeloid progenitors and cooperates with BCR-ABL1 to induce a CML blast crisis-like disease in mice, the hypothesis driving this proposal is that aberrant SETBP1 expression significantly contributes to persistence of TKI-resistant Ph+ B-ALL LICs. Thus, the overall objective of this proposal is two-fold: a) understand the requirement of the SETBP1-PP2A interplay for Ph+ B-ALL LIC self-renewal/survival, and b) assess the therapeutic relevance of SETBP1 downregulation and pharmacologic restoration of PP2A activity against TKI- resistant LICs. Specifically, we will: 1) determine whether PP2A is inhibited in Ph+ B-ALL LICs and, if so, assess whether SETBP1 is part of the PP2A inhibitory complex; and 2) investigate the effects of SETBP1 downmodulation and PAD (e.g. OSU2S, FTY720) treatment on Ph+ B-ALL LIC survival/self-renewal by colony- forming cell/replating and serial BM transplantation assays. We are confident that the successful completion of this work will not only advance our knowledge on the role of SETBP1 in leukemias but based on the discoveries we made in the past few years, will also facilitate new observations in the field of Ph+ B-ALL and that some of them will reveal new PAD-based strategies for therapeutic intervention. Hence, the strong importance and high relevance of this work for basic and translational cancer research.